Project Summary Hematopoietic stem cell (HSC) transplantation has been used as a standard treatment for a number of hematopoietic disorders and malignancies to achieve blood regeneration in many patients. However, procuring a sufficient number of compatible HSC sources for patients who need allogeneic transplantation remains challenging, and thus only a fraction of the patients undergo transplantation. Therefore, new advances are needed to develop strategies to either expand HSCs ex vivo or regenerate HSCs in vivo. Multipotent bone marrow (BM) mesenchymal stromal cells (MSCs) are the major constituents of the HSC niche in the bone marrow, providing key regulatory signals to program hematopoiesis. How MSCs can be harnessed remains an important goal to achieve clinical benefits for blood regeneration. Advances in the field of biomechanics have revealed that a range of matrix stiffness exhibited by the native BM milieu elicits profound contractile force- dependent biological responses in MSCs. The goal of this proposal is thus to evaluate the potential of biomaterials with tunable matrix stiffness for use in controlling MSCs to facilitate blood regeneration. The overall hypothesis is that matrix stiffness controls secretion of proteins from MSCs, and in turn, influences hematopoiesis in a paracrine manner. The specific aims include: (1) Elucidate mechanisms behind matrix stiffness-dependent release of hematopoietic factors from MSCs. (2) Engineer injectable hydrogel microdroplets that can encapsulate single MSCs with tunable matrix stiffness. (3) Assess the therapeutic effect of MSCs in hydrogel microdroplets on human blood regeneration in vivo. During the K99 training period, this award will be used to further the candidate's training in stem cell mechanobiology, and for him to become competent in physical approaches to biomaterial design and advanced microtechnologies. This research will be conducted in a major bioengineering laboratory at Harvard University that focus on biomaterial-based technology development for tissue engineering and studying mechanotransduction, with close clinical connections at Massachusetts General Hospital. This experience will ensure a smooth transition into the R00 phase for the candidate to become an independent translational investigator at the interface between mechanobiology and bioengineering to develop novel therapeutic strategies for hematopoietic disorders.